The invention relates to a method for identification of substances which are applicable for treatment or prevention of an insufficient longitudinal growth of the eye (hypermetropia) or for treatment or prevention of an excessive longitudinal growth of the eye (myopia); substances identified by the method for treating or preventing conditions related to the longitudinal growth of the eye; substances and mixtures of substances for the preparation of a pharmaceutical composition for the treatment or prevention of abnormal growth of the axial length of the eye.
Myopia is caused by the length of the eye being too big in relation to the optical strength of the cornea and the lens so that the picture of a distant object is focused in a point in front of the retina, whereas the picture produced on the retina will be blurred. In other words, myopia is caused by an anomaly between the length of the eye (the axial length) and the refraction in the cornea and the lens.
The longitudinal growth of the eye (from approximately 17 mm at birth to approximately 24 mm) during the childhood is caused by expansion of the eye content and thus stretching the immature connective tissue in the sclera of the eye which adjusts to the new size of the eye. Normally the eye will reach its permanent length at the age of 12, at which time the connective tissue in the sclera reaches an appropriate degree of maturity and the longitudinal growth of the eye will stop.
In myopic persons the longitudinal growth of the eye is too high and the longitudinal growth of the eye continues for a longer period of time than in normal individuals. Hypermetropia is caused by the length of the eye being too short in relation to the optical strength of the cornea and the lens. Hypermetropia usually prevails at birth and is normally recorded at the age of 3-5 years. Subsequently it will reduce in proportion with the growth of the eye until the age of 12, from which age it will remain constant for the rest of the persons life.
Approximately 25%, of the population are myopic. In some myopic persons the axial length is normal (physiological myopia, of  less than xe2x88x924 dioptry), in other persons, the axial length grows from the age of 8-10 unproportionally much until the approximate age of 20, and subsequently the axial length and thus the myopia are stable (intermediary myopia, glass strength of from approximately xe2x88x924 to xe2x88x926 dioptries).
Finally, in rare cases a continuously growing axial length throughout the entire life can be seen, often connected with bulges in weak areas of the eye wall (scleraectasies). Here the myopia can reach extreme levels for glass strengths of up to approximately xe2x88x9240 dioptries (Excessive/pathological myopia).
The intermediary form, and the excessive one in particular, is connected to a high risk of severe sight threatening complications such as e.g. retinal detachment, degenerative changes in the yellow spot of the eye (macula degeneration) and glaucoma.
In the Western part of the world, severe myopia is among the most important causes of blindness.
The group of myopic persons with a glass strength of more than xe2x88x926, which comprises parts of the intermediary group and the entire excessive group, comprises approximately 2% of the population, e.g. in Denmark approximately 100 000 persons (Curtin, B. J.: The myopys: Basic science and clinical management, Harper and Row, Philadelphia, (1985)).
The cause of axial length conditional myopia is unknown.
It is however known that the longitudinal growth of the eye can be increased by disturbance of the image formation on the retina, eg. experimentally by sewing together the eye lids of test animals (visual deprivation) (Yinon, U., Current Eye Research, vol. 3, 4, 677-690, 1984).
Administration of dopaminergic substances (apomorphine) in test animals exposed to visual deprivation inhibits the development of myopia. (Iuvone, P. M., Invest. Ophthalmol., Vis. Sci., 32, 1674-77 (1991)).
U.S. Pat. No. 5,055,302, Laties and Stone, shows a method for control of abnormal postnatal growth of the eye of an animal with the application of vasoactive intestinal peptide (VIP), PH1 or analogues of such peptides. Such peptides were found to restrain the axial longitudinal growth of a myopic eye.
U.S. Pat. No. 5,122,522, Laties and Stone, shows a method for control of abnormal postnatal growth of the eye of an animal with the application of pirenzepine, an anticholinergic substance (M1 Muscarine antagonist). The axial longitudinal growth was inhibited by administration of pirenzepine.
PCT-patent application publication No. WO 94/25034, Laties and Stone, shows a method for control of abnormal postnatal growth of the eye of an animal with the application of tricyclical-substances (antidepressiva). The axial longitudinal growth was inhibited by administration of tricyclical substances.
However, in most cases myopia and hypermetropia are benign conditions which can easily be corrected by means of glasses. In order to justify a medical treatment of these conditions, such treatment must be effective at relatively low dosages and roughly without any side effects, accordingly, as application of VIP, dopaminergic anticholineric or tricyclical substances is connected to risk of side effects as, simultaneously, the substances have considerable psychochemical effects these prior art substances are not suitable for such treatment.
It is also a theory that the growth of the eye can be.caused by passive stretching of the scleral connective tissue (Norton, T. T., Invest. Ophthalmol. Vis. Sci., 37(3), S324 (1996), Siegwart Jr., J. T., Invest. Ophthalmol. Vis. Sci., 37(3), S324 (1996)). Thus it is been shown possible to trigger irreversible stretching of the sclera in young rabbits by increasing the intraoccular pressure but it is not possible to stretch the sclera in mature rabbits (Greene, P. R., ARVO Abstracts, 1978, p. 297). However, tests with reduction of the intraoccular pressure by means of beta-blocking eye drops in humans developing myopia have no effect (Jensen, H., Acta, Ophthalmol., Suppl. 200, 69 (1991)).
There is no model for animal experiments which precisely corresponds to the human conditions. As mentioned above, it is possible to provoke myopia in some animals, e.g. cats and chicken, by sewing together the eye lids of newborn animals, but partly this experimental myopia develops much more rapidly than in humans, and partly the biological age of the animal (newborn) does not correspond to the same age when the myopia typically occurs in human (8-12 years). Furthermore, in chicken the sclera is considerably anomalous as it partly consists of cartilage.
As the conditions of the eye related to the refractory system is extremely common and preventive treatment is to be applied to children, probably during years of treatment, effective substances should be very safe. Accordingly, it would be a considerable improvement if a method for identification of a number of substances having an effect on the longitudinal growth of the eye was available. Among substances such identified, it would subsequently be possible to select, appropriate substances characterized by high efficiency and few side effects.
The present invention is related to methods for identification of substances or groups of substances being candidates for the treatment or prevention of disease of the eye related to the longitudinal growth of the eye.
One of the methods for identification of effective substances according to the present invention is related to the fact that, developmentwise, the retinal pigment epithelium is a part of the retina and forms an electrochemically active cell layer which is located between the choroid membrane of the eye and the neuronal part of the retina (neuroretina). It forms an electrically tight barrier and due to active ion transport (based on the Na+-K+ pump) it creates a difference in potential (the standing potential), the cornea-fundal potential, between the inner and the outer part of the eye of approximately 5 Mv.
Due to the anatomical conditions of the eye, this electrical field is in the nature of a dipole with + at the cornea and xe2x88x92 at the back pole of the eye. The size and changes of the standing potential can thus be estimated with electrodes placed on each side of the eye by means of sideway movements of the eye. (EOG examination (electroocculography which is well known in the art).
The standing potential is furthermore positively correlated with the size of the socalled c-wave in ERG-recordings (electroretinography, which is well known in the art). According to one aspect of the present invention, the growth of the eye is related to the size and changes of the standing potential of the retinal pigment epithelium of the eye.
The transepithelial potential of the retinal pigment epithelium is maintained by a difference in ionic concentration between the cytoplasm of the pigment epithelial cell and the internal and external-surface. The state of the Ca2+-channels determines intracellular Ca2+. According to one aspect of the present invention, it is believed that substances affecting the state of Ca2+ simultaneously affect the standing potential created by the retinal pigment epithelium. The state of the Ca2+-channels may be determined by [3H] ryanodine binding as the plant alkaloid ryanodine has been extensively used to study the functional interaction of the Ca2+-release channel (Meissner G: Ryanodine receptor/Ca2+ release channels and their regulation by endogenous effectors, Annu Rev Physiol 1994;56:485-508).
Accordingly, in a further aspect of the invention, the effect of different drugs on [3H]-ryanodine binding can be used to screen for drugs that either strengthens the scleral connective tissue (increases the content of proteoglycans in sclera), or weakens it (decreases the content of proteoglycanes in sclera).
According to a still further theory behind the present invention, the biomechanical strength of the tissue is believed to be decisive for the stretching of the sclera rather than a change of the intraoccular pressure as described in the prior art above. Substances increasing or decreasing the strength of the scleral tissue may be used according to the present invention for the treatment or prevention of disease relating to the longitudinal growth of the eye. In a further aspect, substances having a substantially selective effect on the posterior part of the sclera is preferred.
In a still further aspect of the present invention, substances having an effect on the content of proteoglycanes in the scleral tissue may be used for the treatment or prevention of disease relating to the longitudinal growth of the eye.
Connective tissue such as eg. sclera mainly consists of scattered cells in a base substance of proteoglycanes (proteins added to glycosaminoglycanes (branched polysaccharides)) and collagen fibres. The biomechanical properties of the tissue are determined by the content of these components and their organisation (Scott, J. E., Dermatan Sulphate Proteoglycans, Portland Press, 1993).
Furthermore, it has been demonstrated that fibroblasts in cell cultures may react to physiological electrical fields by taking on an oblong shape and orienting itself with the longitudinal axis orthogonally with the direction of the field (Erickson, C. A, et al., Cell Biol, Vol. 98, January 1984, 296-307). It has also been demonstrated that the connective tissue substance which is produced by fibroblasts may be determined by the shape of the cell (Evangilisti, R. et al, Eur. J. Histochem., 37, 161-172, 1993).
The present invention relates to a method for identification of substances which have an effect, either inhibiting or increasing, on the longitudinal growth of the human eye.
In another aspect, the invention relates to a method for treating and/or preventing myopia and hypermetropia and to pharmaceutical compositions for such treatment, as well as for use of substances for the preparation of medicaments useful for such treatment or prevention.
According to the present invention it is presumed that some of these substances act mainly on the retinal pigment epithelium and in addition on the receptors of the neuroretina.
In accordance with the present invention it is however believed, that the substances in tha last end excerts the treating or preventing effect through an effect on the ion transport in and out of the cell in the retinal pigment epithelium and thereby exerts its effect on the longitudinal growth of the eye.
In one aspect the invention comprises screening of an optional number of substances for the effect on the metabolical activity in the retinal pigment epithelium of the eye.
This effect may be measured by different methods. One of these methods relates to the activation of the Ca2+-release channel. This Ca2+-release channel may be influenced by different effectors on receptors on the cell membrane. Examples of such receptors are the ryanodine receptor (RyR) and the inositol triphosphate (IP3) receptor.
In another aspect, the present invention relates to the surprising finding that substances affecting the size of the standing potential are useful for the treatment or prevention of abnormal growth of the axial length of the eye.
Accordingly, by measuring of effect of the substances on the electrochemical potential over the retinal pigment epithelium, the so-called xe2x80x9cstanding potentialxe2x80x9d, or the effect on the so-called xe2x80x9cc-wavexe2x80x9d by electro retinography (ERG), suitable substances for treating or preventing conditions related to the longitudinal growth of the eye are easily identified.
Substances increasing the standing potential or c-wave will inhibit the longitudinal growth of the eye and will thus be applicable for treatment of myopia. Substances reducing the standing potential or c-wave will increase the longitudinal growth of the eye and will thus be applicable for treatment of hypermetropia.
In a further aspect of the invention it is believed that physiological electrical fields are important in relation to the extracellular matrix of the scleral tissue and thereby to the biomechanical properties of sclera. An increase of the electrical field is believed to influence the connective tissue cells in the sclera to take on an oblong form and deposit orthogonally on the field, i.e. in the longitudinal direction of the sclera, making the tissue organise more appropriately as concerns content of base substance and packing of collagen fibres. After long time treatment the sclera will be more resistant to draft and thus less likely to give in to the intraoccular pressure, thus avoiding development of myopia.
In a further aspect of the invention, substances affecting the composition of the proteoglycanes may be used for the treatment of abnormalities of the longitudinal growth of the eye.
In a further aspect, the invention comprises a method involving analysis of sclera tests from test animals after treatment (0-6 months) with substances influencing the sclera. Accordingly, the method comprises identifying one or more of the following elements: the effect of the substance on the proteoglycanes; the distribution between various glycosaminoglycane types; and the content of collagen specific amino acids. The analysis may additionally comprise identifying the density of collagen fibrils and the distribution between various fibrils diameters by means of electron microscopy.
According to the present invention, it is possible that the effect on the composition of the proteoglycanes and/or the collagen specific amino acid present in the connective tissue of the sclera of the eye is due to an influence on the metabolical activity in the retinal pigment epithelium of the substance of the eye.
Substances increasing the content of proteoglycanes and collagen specific amino acids will strengthen the connective tissue in the sclera, reduce the longitudinal growth of the eye and thus work against myopia. Substances increasing the content of dermatane sulphate in proportion to the other glycosaminocglycanes will furthermore strengthen the connective tissue in the sclera and work against myopia. Substances increasing the density of collagen fibrils and increasing the diameter of the fibrils will furthermore strengthen the connective tissue in the sclera and work against myopia.
Substances reducing the content of proteoglycanes and collagen specific amino acids will weaken the connective tissue in the sclera, increase the longitudinal growth of the eye and thus work against hypermetropia. Substances reducing the content of dermatane sulphate in proportion to the other glycosaminoglycanes will weaken the connective tissue in the sclera and work against hypermetropia. Substances reducing the density of collagen fibrils and reducing the diameter of the fibrils will furthermore weaken the connective tissue in the sclera and work against hypermetropia.
In a further aspect, the invention relates to a method for identifying substances affecting the composition of the proteoglycanes of the sclera of the eye, and thereby exert an effect of the strength of the connective tissue.